A longstanding goal in biomedical research has been to create organotypic co-cultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized 3D co-culture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D co-culture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells. This study represents the first effort to combine these particular cell types into an organized bronchiole co-culture. These cell layers were first cultured in 3D by magnetic levitation and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin & eosin staining of the resulting co-culture showed four distinct layers within the 3D co-culture. Immunohistochemistry confirmed the phenotype of each of the four cell types, and showed organized extracellular matrix formation, particularly with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle α-actin, vimentin, and fibronectin demonstrate the maintenance of phenotype for endothelial cells, smooth muscle cells, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% FBS showed that epithelial cells maintained phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D co-cultures that maintain phenotype and induce extracellular matrix formation.